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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Dopamine modulation of prefrontal cortex activity is manifold and operates at multiple temporal and spatial scales

While the function of dopamine in subcortical structures is largely limited to reward and movement, dopamine neurotransmission in the prefrontal cortex (PFC) is critical to a multitude of temporally and functionally diverse processes such as attention, working memory, behavioral flexibility, action selection, and stress adaptation. How does dopamine influence PFC computation of multiple temporally diverse functions? Here we find causation between sustained and burst patterns of phasic dopamine neuron activation and contemporaneous modulation of PFC neuronal activity at multiple spatio-temporal scales. These include a multidirectional and weak impact on individual PFC neuron rate activity and a robust influence on coordinated ensemble activity, gamma oscillations, and gamma-theta coupling that persisted for minutes. In addition, PFC network responses to burst pattern of dopamine firing were selectively strengthened in behaviorally active states. Thus, dopamine modulation of PFC is spatiotemporally diverse and is dictated by the pattern of dopamine neuron activation and behavioral state. These findings provide insight on the multiplex pattern of modulation by dopamine that may influence PFC computation of temporally diverse functions.

neuroscience↗

bioRxiv2018

Genomic differentiation is initiated without physical linkage among targets of divergent selection in Fall armyworms

The process of speciation involves whole genome differentiation by overcoming gene flow between diverging populations. We have ample knowledge which evolutionary forces may cause genomic differentiation, and several speciation models have been proposed to explain the transition from genetic to genomic differentiation. However, it is still unclear what are critical conditions enabling genomic differentiation in nature. The Fall armyworm, Spodoptera frugiperda, is observed as two sympatric strains that have different host-plant ranges, suggesting the possibility of ecological divergent selection. In our previous study, we observed that these two strains show genetic differentiation across the whole genome with an unprecedentedly low extent, suggesting the possibility that whole genome sequences started to be differentiated between the strains. In this study, we analyzed whole genome sequences from these two strains from Mississippi to identify critical evolutionary factors for genomic differentiation. The genomic Fst is low (0.017) while 91.3% of 10kb windows have Fst greater than 0, suggesting genome-wide differentiation with a low extent. We identified nearly 400 outliers of genetic differentiation between strains, and found that physical linkage among these outliers is not a primary cause of genomic differentiation. Fst is not significantly correlated with gene density, a proxy for the strength of selection, suggesting that a genomic reduction in migration rate dominates the extent of local genetic differentiation. Our analyses reveal that divergent selection alone is sufficient to generate genomic differentiation, and any following diversifying factors may increase the level of genetic differentiation between diverging strains in the process of speciation.

evolutionary biology↗

bioRxiv2018

Directed evolution of split APEX peroxidase

APEX is an engineered peroxidase that catalyzes the oxidation of a wide range of substrates, facilitating its use in a variety of applications, from subcellular staining for electron microscopy to proximity biotinylation for spatial proteomics and transcriptomics. To further advance the capabilities of APEX, we used directed evolution to engineer a split APEX tool (sAPEX). Twenty rounds of FACS-based selections from yeast-displayed fragment libraries, using three different yeast display configurations, produced a 200-amino acid N-terminal fragment (with 9 mutations relative to APEX2) called \"AP\" and a 50-amino acid C-terminal fragment called \"EX\". AP and EX fragments were each inactive on their own but reconstituted to give peroxidase activity when driven together by a molecular interaction. We demonstrate sAPEX reconstitution in the mammalian cytosol, on engineered RNA motifs within telomerase noncoding RNA, and at mitochondria-endoplasmic reticulum contact sites.

bioengineering↗

bioRxiv2018

Actomyosin-driven force patterning controls endocytosis at the immune synapse

An important channel of cell-to-cell communication is direct contact. The immune synapse is a paradigmatic example of such type of interaction: it forms upon engagement of antigen receptors in lymphocytes by antigen-presenting cells and allows the local exchange of molecules [1]. Although [2], how forces organize and mechanics has been shown to play an important role in this process impact on synapse function is unknown. We found that mechanical forces are spatio-temporally patterned at the immune synapse: global contractile forces are observed at the synapse periphery and local point-like forces are detected at its centre. The global contractile forces result from a pulsatile centripetal actomyosin flow that leads to formation of F-actin protrusions from which the central point like forces emerge. Noticeably, these force-producing actin protrusions constitute the main site of antigen extraction and endocytosis. Accordingly, deletion of the myosin IIA gene leads to impaired B cell responses. The interplay between global and local forces governed by the actomyosin cytoskeleton therefore controls the endocytic function of the immune synapse and might constitute a more general mechanism in the physical regulation of cell-cell interactions.

cell biology↗

bioRxiv2018

cTAGE5 acts as a Sar1 GTPase regulator for collagen export

Secretory proteins synthesized within the endoplasmic reticulum (ER) are exported via coat protein complex II (COPII)-coated vesicles. The formation of the COPII-coated vesicles is initiated by activation of the small GTPase, Sar1. cTAGE5 directly interacts with a guanine-nucleotide exchange factor (GEF), Sec12, and a GTPase-activating protein (GAP) of Sar1, Sec23. We have previously shown that cTAGE5 recruits Sec12 to the ER exit sites for efficient production of activated Sar1 for collagen secretion. However, the functional significance of the interaction between cTAGE5 and Sec23 has not been fully elucidated. In this study, we showed that cTAGE5 enhances the GAP activity of Sec23 toward Sar1. In addition, the interaction of cTAGE5 with Sec23 is necessary for collagen exit from the ER. Our data suggests that cTAGE5 acts as a Sar1 GTPase regulator for collagen secretion.

cell biology↗

bioRxiv2018

Effects of inflammation-induced oxidation on the dimerization of fibrinogen αC domain

Upon activation, fibrinogen is converted to insoluble fibrin, which assembles into long strings called protofibrils. These aggregate laterally to form a fibrin matrix that stabilizes a blood clot. Lateral aggregation of protofibrils is mediated by the C domain, a partially structured fragment located in a disordered region of fibrinogen. Polymerization of C domains links multiple fibrin molecules with each other enabling the formation of thick fibrin fibers and a fibrin matrix that is stable but can also be digested by enzymes. How-ever, oxidizing agents produced during the inflammatory response have been shown to cause thinner fibrin fibers resulting in denser clots, which are harder to proteolyze and pose the risk of deep vein thrombosis and lung embolism. It has been postulated that oxidation of Met476 located within the C domain hinders its ability to polymerize disrupting the lateral aggregation of protofibrils and leading to the observed thinner fibers. How C domains assemble into polymers is still unclear and yet this knowledge would shed light on the mechanism through which oxidation weakens the lateral aggregation of protofibrils. This study used temperature replica exchange molecular dynamics simulations to investigate the C-domain dimer and how this is affected by oxidation of Met476. The results suggest that multiple binding modes between two alphaC domains can occur and that oxidation decreases the likelihood of dimer formation. Furthermore, the side chain of Met476 acts as a docking spot between C domains and this function is abrogated by its conversion to methionine sulfoxide.

biochemistry↗

bioRxiv2018

Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

Human mitochondrial ribonuclease P (mtRNase P) is an essential three protein complex that catalyzes the 5 end maturation of mitochondrial precursor tRNAs (pre-tRNAs). MRPP3 (Mitochondrial RNase P Protein 3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl methionine (SAM)-dependent methyltransferase MRPP1/2 sub-complex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo. Consistent with this, we found that addition of the MRPP1 co-factor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process and methylate human mitochondrial pre-tRNAs.

biochemistry↗

bioRxiv2018

Orbitofrontal neuron ensembles contribute to inhibitory control

Stopping, or inhibition, is a form of self-control that is a core part of adaptive behavior. We hypothesize that inhibition commands originate, in part, from the orbitofrontal cortex (OFC). We recorded activity of OFC neurons in macaques performing a stop signal task. Decoding analyses revealed a clear difference in ensemble responses that distinguish successful from failed inhibition that begins after the stop signal and before the stop signal reaction time. We also found a different and unrelated ensemble pattern that distinguishes successful from failed stopping before the beginning of the trial. These signals were distinct from, and orthogonal to, value encoding, which was also observed in these neurons. The timing of the early and late signals was, respectively, consistent with the idea that OFC contributes both proactively and reactively to inhibition. These results support the view, inspired by anatomy, that OFC gathers diverse sensory inputs to compute early-stage executive signals.

neuroscience↗

bioRxiv2018

ILEX PARAGUARIENSIS, EXERCISE AND CARDIOPROTECTION: A retrospective analysis

Studies on strategies to generate cardioprotective effects have been on the rise. Previous work by our group with an ex vivo model of ischemia/reperfusion has shown that both the short-term consumption of yerba mate and exercise can each induce protection of cardiac function independently. Surprising, the two strategies together do not, with an apparent loss of their respective cardioprotection activity. To improve our understanding of the mechanisms involved without reperforming the experiments, we have conducted a retrospective data science-analysis that have produced new insights. The analysis shows that yerba mate generated reductive stress. Alone, this stress increased redox damage in the heart that appears to have led to a protective conditioning. In combination with exercise, the effects of mate inhibited the intermittent ROS generation promoted by exercise alone, which diminished the adaptive response in the heart. These results suggest that an understanding of the molecular mechanisms involved with the yerba mate-promoted reductive stress in cardiac tissue could lead to improved strategies to induce cardioprotection.

biochemistry↗

bioRxiv2018

Anti-tumor effects of antimicrobial peptides, targets of the innate immune system, against hematopoietic tumors in Drosophila mxc mutants

The innate immune response is the first line of defense against microbial infections. In Drosophila, three immune pathways induce the synthesis of antimicrobial peptides (AMPs) in the fat body. Recently, it has been reported that certain cationic AMPs exhibit selective cytotoxicity against human cancer cells. However, little is known about their anti-tumor effects. Drosophila mxcmbn1 mutants exhibit malignant hyperplasia in a larval hematopoietic organ called the lymph gland (LG). Here, using RNA-Seq analysis, we found that many immunoresponsive genes, including AMP genes, were up-regulated in the mutants. Down-regulation of these pathways by either a Toll or an imd mutation enhanced the tumor phenotype of the mxc mutants. Conversely, ectopic expression of each of five different AMPs in the fat body significantly suppressed the LG hyperplasia phenotype in the mutants. Thus, we propose that the Drosophila innate immune system can suppress progression of hematopoietic tumors by inducing AMP gene expression. Overexpression of any one of these five AMPs resulted in enhanced apoptosis in the mutant LGs, while no apoptosis signals were detected in controls. We observed that two AMPs, Drosomycin and Defensin, were taken up by circulating hemocyte-like cells, which were associated with LG regions showing reduced cell-to-cell adhesion in the mutants; another AMP, diptericin, was directly localized on the tumors without intermediating hemocytes. These results lead us to conclude that the AMPs have a specific cytotoxic effect that enhance apoptosis exclusively in the tumor cells.\n\nSummary statementAntimicrobial peptides can be associated with tumor cells generated in a hematopoietic tissue in Drosophila mxc mutants and have an anti-tumor effect in suppressing their growth.

developmental biology↗

bioRxiv2018

Split Selectable Markers

Selectable markers are widely adopted in transgenesis and genome editing for selecting engineered cells with desired genotype but are limited in choices. We present here split selectable markers each allowing for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR/Cas-mediated biallelic knock-ins. Future development of split selectable markers may support enrichment or selection of "hyper-engineered" cells containing tens of transgenes or genetic modifications.

bioengineering↗

bioRxiv2018

Hippocampal theta bursting and waveform shape reflect CA1 spiking patterns

Brain rhythms are nearly always analyzed in the spectral domain in terms of their power, phase, and frequency. While this conventional approach has uncovered spike-field coupling, as well as correlations to normal behaviors and pathological states, emerging work has highlighted the physiological and behavioral importance of multiple novel oscillation features. Oscillatory bursts, for example, uniquely index a variety of cognitive states, and the nonsinusoidal shape of oscillations relate to physiological changes, including Parkinsons disease. Open questions remain regarding how bursts and nonsinusoidal features relate to circuit-level processes, and how they interrelate. By analyzing unit and local field recordings in the rodent hippocampus, we uncover a number of significant relationships between oscillatory bursts, nonsinusoidal waveforms, and local inhibitory and excitatory spiking patterns. Bursts of theta oscillations are surprisingly related to a decrease in pyramidal neuron synchrony, and have no detectable effect on firing sequences, despite significant increases in neuronal firing rates during periods of theta bursting. Theta burst duration is predicted by the asymmetries of its first cycle, and cycle asymmetries relate to firing rate, synchrony, and sequences of pyramidal neurons and interneurons. These results provide compelling physiological evidence that time-domain features, of both nonsinusoidal hippocampal theta waveform and the theta bursting state, reflects local circuit properties. These results point to the possibility of inferring circuit states from local field potential features in the hippocampus and perhaps other brain regions with other rhythms.

neuroscience↗

bioRxiv2018

Asymmetric dispersal is a critical element of concordance between biophysical dispersal models and spatial genetic structure in Great Barrier Reef corals

AimWidespread coral bleaching, crown-of-thorns seastar outbreaks, and tropical storms all threaten foundational coral species of the Great Barrier Reef, with impacts differing over time and space. Yet, dispersal via larval propagules could aid reef recovery by supplying new settlers and enabling the spread of adaptive variation among regions. Documenting and predicting spatial connections arising from planktonic larval dispersal in marine species, however, remains a formidable challenge.\n\nLocationThe Great Barrier Reef, Australia\n\nMethodsContemporary biophysical larval dispersal models were used to predict longdistance multigenerational connections for two common and foundational coral species (Acropora tenuis and Acropora millepora). Spatially extensive genetic surveys allowed us to infer signatures of asymmetric dispersal for these species and evaluate concordance against expectations from biophysical models using coalescent genetic simulations, directions of inferred gene flow, and spatial eigenvector modelling.\n\nResultsAt long distances, biophysical models predicted a preponderance of north to south connections and genetic results matched these expectations: coalescent genetic simulations rejected an alternative scenario of historical isolation; the strongest signals of inferred gene flow were from north to south; and asymmetric eigenvectors derived from north to south connections in the biophysical models were significantly better predictors of spatial genetic patterns than eigenvectors derived from symmetric null spatial models.\n\nMain conclusionsResults are consistent with biophysical dispersal models yielding approximate summaries of past multigenerational gene flow conditioned upon directionality of connections. For A. tenuis and A. millepora, northern and central reefs have been important sources to downstream southern reefs over the recent evolutionary past and should continue to provide southward gene flow. Endemic genetic diversity of southern reefs suggests substantial local recruitment and lack of long distance gene flow from south to north.

ecology↗

bioRxiv2018

A model of ganglion axon pathways accounts for percepts elicited by retinal implants

Retinal prostheses, now implanted in over 250 patients worldwide, electrically stimulate surviving cells in order to evoke neuronal responses that are interpreted by the brain as visual percepts ( phosphenes). However, instead of seeing focal spots of light, current implant users perceive highly distorted phosphenes that vary in shape both across subjects and electrodes. We characterized these distortions by asking users of the Argus retinal prosthesis system (Second Sight Medical Products) to draw elicited percepts on a touchscreen. We found that phosphene shape could be accurately predicted by simulating the topographic organization of nerve fiber bundles in each subjects retina. Our model shows that activation of ganglion axons contributes to a rich repertoire of phosphene shapes, successfully replicating percepts ranging from blobs to oriented streaks and wedges depending on electrode location. This work provides a first step towards future devices that incorporate stimulation strategies tailored to each individual patients retinal neurophysiology.\n\nOne Sentence SummaryWe show that the perceptual experience of retinal implant users can be accurately predicted using a computational model that simulates the topographic organization of each individual patients retinal ganglion axon pathways.

neuroscience↗

bioRxiv2018

Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs, which are packaged into virions during the infections of vertebrate and invertebrate cells. However, the roles that the noncapped genomic RNAs (ncgRNAs) play during alphaviral infection have yet to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral infection was assessed by using mutants of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the proteins capping efficiency. Specifically, point mutants at residues Y286A and N376A decreased capping efficiency, while a point mutant at D355A increased the capping efficiency of the SINV genomic RNA during genuine viral infection. Viral growth kinetics were significantly reduced for the D355A mutant relative to wild type infection, whereas the Y286A and N376A mutants showed modest decreases in growth kinetics. Overall genomic translation and nonstructural protein accumulation was found to correlate with increases and decreases in capping efficiency. However, genomic, minus strand, and subgenomic viral RNA synthesis was largely unaffected by the modulation of alphaviral capping activity. In addition, translation of the subgenomic vRNA was found to be unimpacted by changes in capping efficiency. The mechanism by which decreased presence of ncgRNAs reduced viral growth kinetics was through the impaired production of viral particles. Collectively, these data illustrate the importance of ncgRNAs to viral infection and suggests that they play in integral role in the production of viral progeny.\n\nImportanceAlphaviruses have been the cause of both localized outbreaks and large epidemics of severe disease. Currently, there are no strategies or vaccines which are either safe or effective for preventing alphaviral infection or treating alphaviral disease. This deficit of viable therapeutics highlights the need to better understand the mechanisms behind alphaviral infection in order to develop novel antiviral strategies for alphaviral disease. In particular, this study details a previously uncharacterized aspect of the alphaviral life cycle, the importance of noncapped genomic viral RNAs to alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging.

microbiology↗

bioRxiv2018

Bacterial T6SS Effector EvpP Inhibits Neutrophil Recruitment via Jnk-Caspy Inflammasome Signaling In vivo

The type VI secretion system (T6SS) comprises dynamic complex bacterial contractile nanomachines and is used by many bacteria to inhibit or kill other prokaryotic or eukaryotic cells. Previous studies have revealed that T6SS is constitutively active in response to various stimuli, or fires effectors into host cells during infection. It has been proposed that the T6SS effector EvpP in Edwardsiella piscicida can inhibit NLRP3 inflammasome activation via the Ca2+-dependent JNK pathways. Here, we developed an in vivo infection model by microinjecting bacteria into the tail vein muscle of 3-day-post-fertilized zebrafish larvae, and found that both macrophages and neutrophils are essential for bacterial clearance. Further study revealed that EvpP plays a critical role in promoting the pathogenesis of E. piscicida via inhibiting the phosphorylation of Jnk signaling to reduce the expression of cxcl8a, mmp13 and IL-1{beta} in vivo. Subsequently, by utilizing Tg (mpo:eGFP+/+) zebrafish larvae for E. piscicida infection, we found that the EvpP-inhibited Jnk-caspy inflammasome signaling axis significantly suppressed the recruitment of neutrophils to infection sites, and the caspy- or IL-1{beta}-MO knockdown larvae were more susceptible to infection and failed to restrict bacterial colonization in vivo.\n\nIMPORTANCEInnate immunity is regulated by phagocytic cells and is critical for host control of bacterial infection. In many bacteria, T6SSs can affect bacterial virulence in certain environments, but little is known about the mechanisms underlying T6SS regulation of innate immune responses during infection in vivo. Here, we investigated the role of an E. piscicida T6SS effector EvpP in manipulating the reaction of neutrophils in vivo. We show that EvpP inhibits the activation of Jnk-caspy inflammasome pathway in zebrafish larvae, and reveal that macrophages are essential for neutrophil recruitment in vivo. This interaction improves our understanding about the complex and contextual role of a bacterial T6SS effector in modulating the action of myeloid cells during infection, and offers new insights into the warfare between bacterial weapons and host immunological surveillance.

microbiology↗

bioRxiv2018

Identifying loci under positive selection in complex population histories

Detailed modeling of a species history is of prime importance for understanding how natural selection operates over time. Most methods designed to detect positive selection along sequenced genomes, however, use simplified representations of past histories as null models of genetic drift. Here, we present the first method that can detect signatures of strong local adaptation across the genome using arbitrarily complex admixture graphs, which are typically used to describe the history of past divergence and admixture events among any number of populations. The method--called Graph-aware Retrieval of Selective Sweeps (GRoSS)--has good power to detect loci in the genome with strong evidence for past selective sweeps and can also identify which branch of the graph was most affected by the sweep. As evidence of its utility, we apply the method to bovine, codfish and human population genomic data containing multiple population panels related in complex ways. We find new candidate genes for important adaptive functions, including immunity and metabolism in under-studied human populations, as well as muscle mass, milk production and tameness in specific bovine breeds. We are also able to pinpoint the emergence of large regions of differentiation due to inversions in the history of Atlantic codfish.

evolutionary biology↗

bioRxiv2018

Characterisation of deubiquitylating enzymes in the cellular response to high-LET ionising radiation and complex DNA damage

PurposeIonising radiation, particular high linear energy transfer (LET) radiation, can induce complex DNA damage (CDD) where two or more DNA lesions are induced in close proximity which contributes significantly to the cell killing effects. However knowledge of the enzymes and mechanisms involved in co-ordinating the recognition and processing of CDD in cellular DNA are currently lacking.\n\nMethods and MaterialsAn siRNA screen of deubiquitylation enzymes was conducted in HeLa cells irradiated with high-LET -particles or protons, versus low-LET protons and x-rays, and cell survival monitored by clonogenic assays. Candidates whose depletion led to decreased cell survival specifically in response to high-LET radiation were validated in both HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells, and the association with CDD repair was confirmed by using an enzyme modified neutral comet assay.\n\nResultsDepletion of USP6 decreased cell survival specifically following high-LET -particles and protons, but not by low-LET protons or x-rays. USP6 depletion caused cell cycle arrest and a deficiency in CDD repair mediated through instability of poly(ADP-ribose) polymerase-1 (PARP-1). This phenotype was mimicked using the PARP inhibitor olaparib.\n\nConclusionUSP6 controls cell survival in response to high-LET radiation by stabilising PARP-1 protein levels which is essential for CDD repair. We also describe synergy between CDD induced by high-LET protons and PARP inhibition in effective cancer cell killing.

cancer biology↗